PROJECT 1 ? BIOLOGY OF DNA DEAMINASES IN BREAST CANCER ABSTRACT Estrogen receptor (ER)-positive breast cancer is the most common form of breast cancer, accounting for over 75% of invasive breast cancers diagnosed each year. The overall mutation landscape in ER-positive breast cancer is multifactorial, but the DNA deaminase APOBEC3B (A3B) accounts for nearly 20% of base- substitution mutations in primary disease and over 50% in metastases. A3B is not expressed in normal mammary epithelial cells and becomes overexpressed in the majority of breast cancers. A3B overexpression correlates with poor clinical outcomes for ER-positive breast cancer, including recurrence, metastasis, and drug resistance. Our Program is testing the overarching hypothesis that A3B inhibition, as an adjuvant to primary treatment options, will help to prevent detrimental mutation-driven outcomes such as drug resistance and metastasis. Project 1 will contribute directly to collaborative Program efforts to test this hypothesis through 3 specific aims. In Aim 1, we propose to develop reporter systems for quantifying A3B-mediated editing in living cells, including an innovative, transportable reporter. In one potential application, this system will enable rapid testing of candidate small molecule A3B inhibitors in a panel of breast cancer cell lines as candidate compounds are developed through the concerted activities of all Program components. In Aim 2, we will delineate mechanisms of protein-level A3B regulation in normal and breast cancer cells. These studies will focus on protein?protein interactions prioritized by proteomics data sets. Comprehensive characterization of direct interactions is also anticipated to reveal potentially druggable surfaces for collaborative studies on chemical probes (Project 2), computational modeling (Core C), and structural biology (Project 3). In Aim 3, we will address how A3B-catalyzed genomic uracil lesions are processed into error-free and mutagenic outcomes by different DNA repair pathways. These studies have the potential to reveal molecular dependencies in DNA repair that are specific to breast tumor cells undergoing elevated levels of DNA damage catalyzed by A3B. Thus, Project 1 is an integral component of this overall Program because it will provide innovative assays for quantifying A3B activity in living breast cancer cells, yield molecular insights into regulatory and potentially druggable protein surfaces, and uncover genetic dependencies that may constitute new opportunities for diagnostic and therapeutic development.